Hepatic interoception in health and disease.

The liver is a large organ with crucial functions in metabolism and immune defense, as well as blood homeostasis and detoxification, and it is clearly in bidirectional communication with the brain and rest of the body via both neural and humoral pathways. A host of neural sensory mechanisms have been proposed, but in contrast to the gut-brain axis, details for both the exact site and molecular signaling steps of their peripheral transduction mechanisms are generally lacking. Similarly, knowledge about function-specific sensory and motor components of both vagal and spinal access pathways to the hepatic parenchyma is missing. Lack of progress largely owes to controversies regarding selectivity of vagal access pathways and extent of hepatocyte innervation. In contrast, there is considerable evidence for glucose sensors in the wall of the hepatic portal vein and their importance for glucose handling by the liver and the brain and the systemic response to hypoglycemia. As liver diseases are on the rise globally, and there are intriguing associations between liver diseases and mental illnesses, it will be important to further dissect and identify both neural and humoral pathways that mediate hepatocyte-specific signals to relevant brain areas. The question of whether and how sensations from the liver contribute to interoceptive self-awareness has not yet been explored.

Innervation pattern of the unclosed detrusor muscle in classic bladder exstrophy: a study of patients with urothelial overexpression of nerve growth factor.

PURPOSE: An overexpression of nerve growth factor (NGF) in the urothelium is discussed to lead to neuronal hyperinnervation of the bladder detrusor. The aim was to assess the sensory and sympathetic innervation of the detrusor in unclosed exstrophic bladders patients with known overexpression of NGF in the urothelium. METHODS: Full-thickness bladder biopsies were prospectively obtained from 34 infants at delayed primary bladder closure between 01/2015 and 04/2020. The bladder biopsies were immunohistochemically stained with antibodies against S100, calcitonin gene-related peptide (anti-CGRP), Neurofilament 200 (anti-NF200), and tyrosine-hydroxylase (anti-TH). Specimens from 6 children with congenital vesicoureterorenal reflux (VUR) served as controls. RESULTS: There was no statistically significant difference in nerve fiber density in any of the immunohistochemical assessments (anti-S100 [p = 0.210], anti-CGRP [p = 0.897], anti-NF200 [p = 0.897]), and anti-TH [p = 0.956]) between patients with BE and patients with VUR. However, we observed a trend toward lower nerve fiber densities in exstrophic detrusor. CONCLUSION: Overall our results showed an unharmed innervation pattern in this cohort but a lower density of nerve fibers in the detrusor compared to controls. Further studies in patients after successful primary closure are needed to clarify the potential impact of the urothelial overexpression of NGF modulating the innervation pattern in exstrophic bladders.

Sensory spinal interoceptive pathways and energy balance regulation.

Interoception plays an important role in homeostatic regulation of energy intake and metabolism. Major interoceptive pathways include gut-to-brain and adipose tissue-to brain signaling via vagal sensory nerves and hormones, such as leptin. However, signaling via spinal sensory neurons is rapidly emerging as an additional important signaling pathway. Here we provide an in-depth review of the known anatomy and functions of spinal sensory pathways and discuss potential mechanisms relevant for energy balance homeostasis in health and disease. Because sensory innervation by dorsal root ganglia (DRG) neurons goes far beyond vagally innervated viscera and includes adipose tissue, skeletal muscle, and skin, it is in a position to provide much more complete metabolic information to the brain. Molecular and anatomical identification of function specific DRG neurons will be important steps in designing pharmacological and neuromodulation approaches to affect energy balance regulation in disease states such as obesity, diabetes, and cancer.

Neurochemical classification of serotonin-immunoreactive neurons co-innervating motor endplates in the mouse esophagus.

Serotonin immunoreactivity was previously found in myenteric neurons co-innervating motor endplates in the mouse esophagus striated muscle and an involvement in motility control was suggested. However, it is not known if other neuroactive substances are present in these neurons and to what extent they co-localize. First, vasoactive intestinal peptide (VIP) was established as a bona fide marker for putative inhibitory myenteric neurons by evaluating co-localization with neuronal nitric oxide synthase (nNOS) and neuropeptide Y (NPY). Then, co-localization of serotonin and VIP was tested in co-innervating axons on motor endplates, which were visualized with α-bungarotoxin (α-BT) by multilabel immunofluorescence. Myenteric ganglia were also surveyed for co-localization in neuronal perikarya and varicosities. nNOS, NPY, and VIP were completely co-localized in enteric co-innervating nerve terminals on motor endplates. After co-staining with VIP, we found (a) serotonin (5-HT)-positive nerve endings without VIP (44% of 5-HT-positively innervated endplates), (b) 5-HT- and VIP-positive endings without co-localization (35%), and (c) 5-HT- and VIP-positive endings with co-localization (21%). About one-fifth of nerve terminals on motor endplates containing 5-HT originate from putative inhibitory peptidegic nitrergic neurons. However, the majority represents a different population presumably subserving different functions.

Functional anatomy of the vagus system: How does the polyvagal theory comply?

Due to its pivotal role in autonomic networks and interoception, the vagus attracts continued interest from both basic scientists and therapists of various clinical disciplines. In particular, the widespread use of heart rate variability as an index of autonomic cardiac control and a proposed central role of the vagus in biopsychological concepts, e.g., the polyvagal theory, provide a good opportunity to recall basic features of vagal anatomy. In addition to the "classical" vagal brainstem nuclei, i.e., dorsal motor nucleus, nucleus ambiguus and nucleus tractus solitarii, the spinal trigeminal and paratrigeminal nuclei come into play as targets of vagal afferents. On the other hand, the nucleus of the solitary tract receives and integrates not only visceral but also somatic afferents.

Functional anatomy of the vagus system - Emphasis on the somato-visceral interface.

Due to its pivotal role in autonomic networks, the vagus attracts continuous interest from both basic scientists and clinicians. In particular, recent advances in vagus nerve stimulation strategies and their application to pathological conditions beyond epilepsy provide a good opportunity to recall basic features of vagal peripheral and central anatomy. In addition to the "classical" vagal brainstem nuclei, i.e., dorsal motor nucleus, nucleus ambiguus and nucleus tractus solitarii, the spinal trigeminal and paratrigeminal nuclei come into play as targets of vagal afferents. On the other hand, the nucleus of the solitary tract receives and integrates not only visceral but also somatic afferents. Thus, the vagus system participates significantly in what may be defined as "somato-visceral interface".

Sympathetic innervation of the mouse kidney and liver arising from prevertebral ganglia.

The sympathetic nervous system (SNS) plays a crucial role in the regulation of renal and hepatic functions. Although sympathetic nerves to the kidney and liver have been identified in many species, specific details are lacking in the mouse. In the absence of detailed information of sympathetic prevertebral innervation of specific organs, selective manipulation of a specific function will remain challenging. Despite providing major postganglionic inputs to abdominal organs, limited data are available about the mouse celiac-superior mesenteric complex. We used tyrosine hydroxylase (TH) and dopamine ß-hydroxylase (DbH) reporter mice to visualize abdominal prevertebral ganglia. We found that both the TH and DbH reporter mice are useful models for identification of ganglia and nerve bundles. We further tested if the celiac-superior mesenteric complex provides differential inputs to the mouse kidney and liver. The retrograde viral tracer, pseudorabies virus (PRV)-152 was injected into the cortex of the left kidney or the main lobe of the liver to identify kidney-projecting and liver-projecting neurons in the celiac-superior mesenteric complex. iDISCO immunostaining and tissue clearing were used to visualize unprecedented anatomical detail of kidney-related and liver-related postganglionic neurons in the celiac-superior mesenteric complex and aorticorenal and suprarenal ganglia compared with TH-positive neurons. Kidney-projecting neurons were restricted to the suprarenal and aorticorenal ganglia, whereas only sparse labeling was observed in the celiac-superior mesenteric complex. In contrast, liver-projecting postganglionic neurons were observed in the celiac-superior mesenteric complex and aorticorenal and suprarenal ganglia, suggesting spatial separation between the sympathetic innervation of the mouse kidney and liver.

Gut-brain communication and obesity: understanding functions of the vagus nerve.

Given the crucial role of the gastrointestinal tract and associated organs in handling nutrient assimilation and metabolism, it has long been known that its communication with the brain is important for the control of ingestive behavior and body weight regulation. It is also clear that gut-brain communication is bidirectional and utilizes both rapid neural and slower humoral mechanisms and pathways. However, progress in understanding these mechanisms and leveraging them for the treatment of obesity and metabolic disease has been hindered by the enormous dimension of the gut mucosa, the complexity of the signaling systems, and lack of specific tools. With the ascent of modern neurobiological technology, our understanding of the role of vagal afferents in gut-brain communication has begun to change. The first function-specific populations of vagal afferents providing nutritional feedback as well as feed-forward signals have been identified with genetics-guided methodology, and it is hoped that extension of the methodology to other neural communication pathways will follow soon. Currently, efficient clinical leveraging of gut-brain communication to treat obesity and metabolic disease is limited to a few gut hormones, but a more complete understanding of function-specific and projection-specific neuronal populations should make it possible to develop selective and more effective neuromodulation approaches.

Murine Esophagus Expresses Glial-Derived Central Nervous System Antigens.

Multiple sclerosis (MS) has been considered to specifically affect the central nervous system (CNS) for a long time. As autonomic dysfunction including dysphagia can occur as accompanying phenomena in patients, the enteric nervous system has been attracting increasing attention over the past years. The aim of this study was to identify glial and myelin markers as potential target structures for autoimmune processes in the esophagus. RT-PCR analysis revealed glial fibrillary acidic protein (GFAP), proteolipid protein (PLP), and myelin basic protein (MBP) expression, but an absence of myelin oligodendrocyte glycoprotein (MOG) in the murine esophagus. Selected immunohistochemistry for GFAP, PLP, and MBP including transgenic mice with cell-type specific expression of PLP and GFAP supported these results by detection of (1) GFAP, PLP, and MBP in Schwann cells in skeletal muscle and esophagus; (2) GFAP, PLP, but no MBP in perisynaptic Schwann cells of skeletal and esophageal motor endplates; (3) GFAP and PLP, but no MBP in glial cells surrounding esophageal myenteric neurons; and (4) PLP, but no GFAP and MBP in enteric glial cells forming a network in the esophagus. Our results pave the way for further investigations regarding the involvement of esophageal glial cells in the pathogenesis of dysphagia in MS.

Vagal mechanisms as neuromodulatory targets for the treatment of metabolic disease.

With few effective treatments available, the global rise of metabolic diseases, including obesity, type 2 diabetes mellitus, and cardiovascular disease, seems unstoppable. Likely caused by an obesogenic environment interacting with genetic susceptibility, the pathophysiology of obesity and metabolic diseases is highly complex and involves crosstalk between many organs and systems, including the brain. The vagus nerve is in a key position to bidirectionally link several peripheral metabolic organs with the brain and is increasingly targeted for neuromodulation therapy to treat metabolic disease. Here, we review the basics of vagal functional anatomy and its implications for vagal neuromodulation therapies. We find that most existing vagal neuromodulation techniques either ignore or misinterpret the rich functional specificity of both vagal efferents and afferents as demonstrated by a large body of literature. This lack of specificity of manipulating vagal fibers is likely the reason for the relatively poor beneficial long-term effects of such therapies. For these therapies to become more effective, rigorous validation of all physiological endpoints and optimization of stimulation parameters as well as electrode placements will be necessary. However, given the large number of function-specific fibers in any vagal branch, genetically guided neuromodulation techniques are more likely to succeed.

Enteric co-innervation of striated muscle in the esophagus: still enigmatic?

The existence of a distinct ganglionated myenteric plexus between the two layers of the striated tunica muscularis of the mammalian esophagus has represented an enigma for quite a while. Although an enteric co-innervation of vagally innervated motor endplates in the esophagus has been suggested repeatedly, it was not possible until recently to demonstrate this dual innervation. Twenty-two years ago, we were able to demonstrate that motor endplates in the rat esophagus receive dual innervation from both vagal nerve fibers originating in the brain stem and from varicose enteric nerve fibers originating in the myenteric plexus. Meanwhile, a considerable amount of data has been gathered on enteric co-innervation and its occurrence in the esophagus of a variety of species including humans, its neurochemistry, spatial relationships on motor endplates, ontogeny and possible functional roles. These data underline the significance of this newly discovered innervation component, although its function in vivo is still largely unknown. The aim of this review, which is an update of our previous paper (Wörl and Neuhuber in Histochem Cell Biol 123(2):117-130. doi: 10.1007/s00418-005-0764-7 , 2005a), is to summarize the current knowledge about enteric co-innervation of esophageal striated muscle and to provide some hints as to its functional significance.

Spatiotemporal Pattern of Human Cortical and Subcortical Activity during Early-Stage Odor Processing.

The dynamics of early-stage cortical and subcortical responses in the human brain to odor stimulation are currently unknown. The objective of the present study was to analyze spatiotemporal patterns of human brain activity during odor perception using magnetoencephalography (MEG). In 12 normosmic healthy subjects, we investigated the onset of brain activity in relation to ipsilateral and contralateral stimulation with 2 odorants. Olfactory stimuli (200ms duration) were applied using an olfactometer, and brain activity was recorded with a 248-magnetometer whole-head MEG system. Olfactory responses were identified shortly (within 150ms) after stimulus onset in both hemispheres. Stimulation on the ipsilateral side yielded signals earlier (starting at 90ms) compared with contralateral stimulation in the primary olfactory cortex, hippocampus, parahippocampal gyrus, amygdala, and orbitofrontal cortex ( P < 0.001). The duration and peak amplitude of olfactory evoked magnetic fields were found to increase with increasing poststimulus time in the majority of the investigated cortical structures ( P ≤ 0.019 and P ≤ 0.021). The study showed the locations of early olfactory brain activity in humans within 150ms after the onset of stimuli. Olfactory activation is processed on the ipsilateral side of stimulation in early stages. After a short delay of 34ms a corresponding pattern of activation was also seen in the contralateral hemisphere, indicating the functional connectivity between the 2 hemispheres in the anterior commissure.

Involvement of catecholaminergic neurons in motor innervation of striated muscle in the mouse esophagus.

Enteric co-innervation is a peculiar innervation pattern of striated esophageal musculature. Both anatomical and functional data on enteric co-innervation related to various transmitters have been collected in different species, although its function remains enigmatic. However, it is unclear whether catecholaminergic components are involved in such a co-innervation. Thus, we examined to identify catecholaminergic neuronal elements and clarify their relationship to other innervation components in the esophagus, using immunohistochemistry with antibodies against tyrosine hydroxylase (TH), vesicular acetylcholine transporter (VAChT), choline acetyltransferase (ChAT) and protein gene product 9.5 (PGP 9.5), α-bungarotoxin (α-BT) and PCR with primers for amplification of cDNA encoding TH and dopamine-ß-hydroxylase (DBH). TH-positive nerve fibers were abundant throughout the myenteric plexus and localized on about 14% of α-BT-labelled motor endplates differing from VAChT-positive vagal nerve terminals. TH-positive perikarya represented a subpopulation of only about 2.8% of all PGP 9.5-positive myenteric neurons. Analysis of mRNA showed both TH and DBH transcripts in the mouse esophagus. As ChAT-positive neurons in the compact formation of the nucleus ambiguus were negative for TH, the TH-positive nerve varicosities on motor endplates are presumably of enteric origin, although a sympathetic origin cannot be excluded. In the medulla oblongata, the cholinergic ambiguus neurons were densely supplied with TH-positive varicosities. Thus, catecholamines may modulate vagal motor innervation of esophageal-striated muscles not only at the peripheral level via enteric co-innervation but also at the central level via projections to the nucleus ambiguus. As Parkinson's disease, with a loss of central dopaminergic neurons, also affects the enteric nervous system and dysphagia is prevalent in patients with this disease, investigation of intrinsic catecholamines in the esophagus may be worthwhile to understand such a symptom.

Vascularization of the dorsal base of the second metacarpal bone: an anatomical study using C-arm cone beam computed tomography.

BACKGROUND: Vascularized bone grafts of the hand are a promising option for treatment of hand abnormalities. Therefore, the purpose of this study was to analyze the arterial anatomy of the dorsal aspect of the second metacarpal base to further investigate this possible donor site for bone grafts. METHODS: The authors examined 16 fresh frozen cadaveric hands by using a C-arm cone beam computed tomography scanner and depicted the three-dimensional course of the second dorsal metacarpal artery and measured the diameter, length, and arc of rotation of this nutritive vessel. In addition, the authors dissected six of the hands under a dissecting microscope and, after selective injection of gelatin dye solution, the authors analyzed the vessel entrances into the bones histologically. RESULTS: In all examined hands, the second dorsal metacarpal artery was a nutritive vessel to the dorsal base of the second metacarpal. The average diameter was 1.3±0.4 mm and the average length of the vascular pedicle was approximately 3.3±0.3 cm. In 14 of 16 cases, the arc of rotation was sufficient to reach the lunate without difficulty. Histologic analysis showed an intrinsic blood supply in the donor region with a vessel diameter of approximately 58 µm. A clinical case with application in Kienböck disease is presented. CONCLUSION: Pedicled vascularized bone grafts from this area are suitable for clinical application to treat Kienböck disease if standard donor sites are unavailable.

Innervation of rat and human dura mater and pericranial tissues in the parieto-temporal region by meningeal afferents.

OBJECTIVE: To reinvestigate the innervation pattern of the dura mater of rat and human middle cranial fossa, the morpho-functional substrate of headache generation, and adjacent extracranial tissues with neuronal in vitro tracing. BACKGROUND: This study was initiated by recent structural and functional findings of meningeal afferent fibers which innervate the cranial dura mater and may project to extracranial tissues. METHODS: Anterograde and retrograde neuronal in vitro tracing was made in formaldehyde fixed hemisected rat and human skulls. The fluorescent tracer DiI was applied to proximally cut meningeal nerves in rat and to distal branches of the spinosus nerve in human calvaria lined by dura mater. After several weeks, the dura mater and deep extracranial tissues were examined with fluorescence microscopy. RESULTS: In addition to a network of meningeal nerve fibers, several fiber bundles were observed, leaving the skull through emissary canals and fissures to innervate the pericranial temporal, parietal, and occipital periosteum. Traced fibers were seen spreading into deep layers of the temporal and upper neck muscles. Retrograde neuronal tracing revealed labeled cell bodies exclusively in the mandibular and maxillary division of the rat trigeminal ganglion, and centrally projecting fibers were identified in the spinal trigeminal tract. Electron microscopy of the cross-sected spinosus nerve showed myelinated and unmyelinated axons with similar numbers in human and rat. CONCLUSIONS: We conclude that a proportion of meningeal afferents innervates extracranial tissues like periosteum and pericranial muscles via collaterals projecting through the skull. These afferents may be nociceptive, some may subserve proprioceptive functions. The finding of extracranial projections of meningeal afferents may be important for our understanding of extracranial impacts on headache generation and therapy.

Changes in calcitonin gene-related peptide (CGRP) receptor component and nitric oxide receptor (sGC) immunoreactivity in rat trigeminal ganglion following glyceroltrinitrate pretreatment.

BACKGROUND: Nitric oxide (NO) is thought to play an important role in the pathophysiology of migraine. Infusion of the nitrovasodilator glyceroltrinitrate (nitroglycerin, GTN), which mobilizes NO in the organism, is an approved migraine model in humans. Calcitonin gene-related peptide (CGRP) is regarded as another key mediator in migraine. Increased plasma levels of CGRP have been found during spontaneous as well as nitrovasodilator-induced migraine attacks. The nociceptive processes and interactions underlying the NO and CGRP mediated headache are poorly known but can be examined in animal experiments. In the present study we examined changes in immunofluorescence of CGRP receptor components (CLR and RAMP1) and soluble guanylyl cyclase (sGC), the intracellular receptor for NO, in rat trigeminal ganglia after pretreatment with GTN. METHODS: Isoflurane anaesthetised rats were intravenously infused with GTN (1 mg/kg) or saline for four hours and two hours later the trigeminal ganglia were processed for immunohistochemistry. Different primary antibodies recognizing CLR, RAMP1, CGRP and sGC coupled to fluorescent secondary antibodies were used to examine immunoreactive cells in serial sections of trigeminal ganglia with epifluorescence and confocal laser scanning microscopy. Several staining protocols were examined to yield optimized immunolabeling. RESULTS: In vehicle-treated animals, 42% of the trigeminal ganglion neurons were immunopositive for RAMP1 and 41% for CLR. After GTN pretreatment CLR-immunopositivity was unchanged, while there was an increase in RAMP1-immunopositive neurons to 46%. RAMP1 and CLR immunoreactivity was also detected in satellite cells. Neurons immunoreactive for sGC were on average smaller than sGC-immunonegative neurons. The percentage of sGC-immunopositive neurons (51% after vehicle) was decreased after GTN infusion (48%). CONCLUSIONS: Prolonged infusion of GTN caused increased fractions of RAMP1- and decreased fractions of sGC-immunopositive neurons in the trigeminal ganglion. The observed alterations are likely immunophenotypic correlates of the pathophysiological processes underlying nitrovasodilator-induced migraine attacks and indicate that signalling via CGRP receptors but not sGC-mediated mechanisms may be enhanced through endogenous NO production.

Extracranial projections of meningeal afferents and their impact on meningeal nociception and headache.

Headaches can be evoked by activation of meningeal nociceptors, but an involvement of pericranial tissues is debated. We aimed to examine a possible extracranial innervation by meningeal afferents in the rat. For in vivo neuronal tracing, dextran amines were applied to the periosteum underlying the temporal muscle. Labeling was observed 2 days later in the parietal dura mater, trigeminal ganglion, and spinal trigeminal nucleus with confocal and electron microscopy. In the hemisected rat head, extracellular recordings were made from meningeal nerve fibers. Release of calcitonin gene-related peptide (CGRP) from the cranial dura mater during noxious stimulation of pericranial muscles was quantified. In vivo capsaicin was injected into the temporal muscle while meningeal blood flow was recorded. In the parietal dura mater, labeled C- and Aδ fibers ramified extensively, accompanied the middle meningeal artery, and passed through the spinosus nerve into the maxillary and mandibular, but not the ophthalmic division of the trigeminal ganglion. Some fibers could be traced into the ipsilateral spinal trigeminal nucleus. Electrophysiological recordings revealed afferent fibers with mechanosensitive receptive fields both in the dura mater and in the parietal periosteum. Noxious stimulation of the temporal muscle caused CGRP release from the dura mater and elevated meningeal blood flow. Collaterals of meningeal nerve fibers project through the skull, forming functional connections between extra- and intracranial tissues. This finding offers a new explanation of how noxious stimulation of pericranial tissues can directly influence meningeal nociception associated with headache generation and why manual therapies of pericranial muscles may be useful in headaches.